Methods: The human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-kappaB activity was examined using a luciferase-based reporter assay and phosphorylation of IkappaBalpha. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection.

Conclusions: S100A4 activates NF-kappaB by inducing phosphorylation of IKKalpha/beta, leading to increased IkappaBalpha phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-kappaB activation and inhibited IKK-mediated phosphorylation of IkappaBalpha. S100A4-induced NF-kappaB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy.

Figure 6: The Ser/Thr kinases MEKK1, NIK and AKT are not involved in S100A4-mediated activation of the IKK complex. A. Representative Western blots of II-11b cells transfected with dominant negative constructs of MEKK1 or NIK (2 μg), stimulated with S100A4 for one hour and analyzed for expression of phosphorylated IκBα. B. Cotransfection of II-11b cells with NF-κB luciferase reporter construct and dominant negative MEKK1 or NIK (2 μg) as described in "Methods". Bars show relative induction of NF-κB activity upon S100A4 treatment compared to relevant control. KD = kinase dead. C. Control experiment to evaluate the ability of the dominant negative MEKK1 and NIK constructs to suppress NF-κB activity. Empty vector was used as transfection control and to adjust the amount of plasmid in each transfection. D. Western blot of II-11b cells treated with 2 μM S100A4 for the indicated time periods, analyzed for levels of phosphorylated AKT and α-tubulin. A, B and D are representative results of three independent experiments.

Mentions:
Previously, we demonstrated JNK phosphorylation after S100A4 treatment of II-11b cells [11]. MEKK1 is a possible common upstream kinase responsible for activating both the IKK complex and JNK [28]. It was therefore of interest to examine whether this kinase could be involved in S100A4-induced activation of NF-κB. However, no significant effect was observed on S100A4-induced IκBα phosphorylation or NF-κB activation when dominant negative MEKK1 was overexpressed (Fig. 6A and 6B). It has also been shown that the Ser/Thr kinases NIK and AKT could be involved in phosphorylation and activation of the IKK complex [29,30]. As for MEKK1, dominant negative NIK was not able to inhibit S100A4-mediated IκBα phosphorylation or NF-κB activation (Fig. 6A and 6B). Wild type MEKK1 and NIK was used in experiments to verify that the dominant negative constructs were able to suppress NF-κB activation induced by MEKK1 or NIK (Fig. 6C). Moreover, AKT phosphorylation at serine residue 473 was unaffected by treatment with S100A4 (Fig. 6D). AKT is normally phosphorylated after PI 3-kinase activation, and the finding that LY294002 had no effect on IκBα phosphorylation strengthens the conclusion that AKT is not involved in S100A4-induced IKK activation.

Figure 6: The Ser/Thr kinases MEKK1, NIK and AKT are not involved in S100A4-mediated activation of the IKK complex. A. Representative Western blots of II-11b cells transfected with dominant negative constructs of MEKK1 or NIK (2 μg), stimulated with S100A4 for one hour and analyzed for expression of phosphorylated IκBα. B. Cotransfection of II-11b cells with NF-κB luciferase reporter construct and dominant negative MEKK1 or NIK (2 μg) as described in "Methods". Bars show relative induction of NF-κB activity upon S100A4 treatment compared to relevant control. KD = kinase dead. C. Control experiment to evaluate the ability of the dominant negative MEKK1 and NIK constructs to suppress NF-κB activity. Empty vector was used as transfection control and to adjust the amount of plasmid in each transfection. D. Western blot of II-11b cells treated with 2 μM S100A4 for the indicated time periods, analyzed for levels of phosphorylated AKT and α-tubulin. A, B and D are representative results of three independent experiments.

Mentions:
Previously, we demonstrated JNK phosphorylation after S100A4 treatment of II-11b cells [11]. MEKK1 is a possible common upstream kinase responsible for activating both the IKK complex and JNK [28]. It was therefore of interest to examine whether this kinase could be involved in S100A4-induced activation of NF-κB. However, no significant effect was observed on S100A4-induced IκBα phosphorylation or NF-κB activation when dominant negative MEKK1 was overexpressed (Fig. 6A and 6B). It has also been shown that the Ser/Thr kinases NIK and AKT could be involved in phosphorylation and activation of the IKK complex [29,30]. As for MEKK1, dominant negative NIK was not able to inhibit S100A4-mediated IκBα phosphorylation or NF-κB activation (Fig. 6A and 6B). Wild type MEKK1 and NIK was used in experiments to verify that the dominant negative constructs were able to suppress NF-κB activation induced by MEKK1 or NIK (Fig. 6C). Moreover, AKT phosphorylation at serine residue 473 was unaffected by treatment with S100A4 (Fig. 6D). AKT is normally phosphorylated after PI 3-kinase activation, and the finding that LY294002 had no effect on IκBα phosphorylation strengthens the conclusion that AKT is not involved in S100A4-induced IKK activation.

Methods: The human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-kappaB activity was examined using a luciferase-based reporter assay and phosphorylation of IkappaBalpha. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection.

Conclusions: S100A4 activates NF-kappaB by inducing phosphorylation of IKKalpha/beta, leading to increased IkappaBalpha phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-kappaB activation and inhibited IKK-mediated phosphorylation of IkappaBalpha. S100A4-induced NF-kappaB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy.